Fermentative preparation process for and crystal forms of cytostatics

ABSTRACT

The invention relates to a new process for concentrating epothilones in culture media, a new process for the production of epothilones, a new process for separating epothilones A and B and a new strain obtained by mutagenesis for the production of epothilones, as well as aspects related thereto. New crystal forms of epothilone B are also described.

[0001] The invention relates to a new biotechnological preparationprocess that can be used on an industrial scale for the production ofepothilones, especially a process for concentrating these compounds inthe culture medium, as well as a new strain for the fermentativepreparation of these compounds. The invention also relates to newcrystal forms of epothilones, especially epothilone B, obtainable by thenew processes, their usage in the production of pharmaceuticalpreparations, new pharmaceutical preparations comprising these newcrystal forms and/or the use of these compounds in the treatment ofproliferative diseases such as tumours, or in the production ofpharmaceutical formulations which are suitable for this treatment.

BACKGROUND TO THE INVENTION

[0002] Of the existing cytotoxic active ingredients for treatingtumours, Taxol® (Paclitaxel; Bristol-Myers Squibb), amicrotubuli-stabilising agent, plays an important role and hasremarkable commercial success. However, Taxol has a number ofdisadvantages. In particular, its very poor solubility in water is aproblem. It therefore became necessary to administer Taxol® in aformulation with Cremophor EL® (polyoxyethylated castor oil; BASF,Ludwigshafen, Germany). Cremophor EL® has severe side effects; forexample it causes allergies which in at least one case have led even tothe death of a patient.

[0003] Although the Taxan class of microtubuli-stabilising anti-canceragents has been commended as “perhaps the most important addition to thepharmaceutical armoury against cancer in the last decade” (see RowinskyE. K., Ann. rev. Med. 48, 353-374 (1997)), and despite the commercialsuccess of Taxol®, these compounds still do not appear to represent areally great breakthrough in the chemotherapy of cancer. Treatment withTaxol® is linked with a series of significant side effects, and a fewprimary classes of compact tumours, namely colon and prostate tumours,respond to this compound only to a small extent (see Rowinsky E. K.,inter alia). In addition, the efficacy of Taxol can be impaired and evencompletely neutralised by acquired resistance mechanisms, especiallythose based on the overexpression of phosphoproteins, which act asefflux pumps for active ingredients, such as “Multidrug Resistance” dueto overexpression of the multidrug transport glycoprotein“P-glycoprotein”.

[0004] Epothilones A and B represent a new class ofmicrotubuli-stabilising cytotoxic active ingredients (see Gerth, K. etal., J. Antibiot. 49, 560-3 (1966)) of the formulae:

[0005] wherein R signifies hydrogen (epothilone A) or methyl (epothiloneB).

[0006] These compounds have the following advantages over Taxol®:

[0007] a) They have better water-solubility and are thus more easilyaccessible for formulations.

[0008] b) It has been reported that, in cell culture experiments, theyare also active against the proliferation of cells, which, owing to theactivity of the P-glycoprotein efflux pump making them “multidrugresistant”, show resistance to treatment with other chemotherapy agentsincluding Taxol® (see Bolag, D. M., et al., “Epothilones, a new class ofmicrotubuli-stabilizing agents with a Taxol-like mechanism of action”,Cancer Research 55, 2325-33 (1995)). And

[0009] c) it could be shown that they are still very effective in vitroagainst a Taxol®-resistant ovarian carcinoma cell line with modifiedβ-tubulin (see Kowalski, R. J., et al., J. Biol. Chem. 272(4), 2534-2541(1997)).

[0010] Pharmaceutical application of the epothilones, for example fortumour treatment, is possible in an analogous manner to that describedfor Taxol, see for example U.S. Pat. Nos. 5,641,803; 5,496,804;5,565,478).

[0011] In order to be able to use the epothilones on a larger scale forpharmaceutical purposes, however, it is necessary to obtain appropriateamounts of these compounds.

[0012] Until now, the extraction of natural substances by means ofmyxobacteria, especially the epothilones from the cell strain SorangiumCellulosum Soce90 (deposited under no. 6773 at the German Collection ofMicroorganisms, see WO 93/10121) was described in literature. In orderto obtain a satisfactory concentration of the natural substances,especially the epothilones, in the culture medium for the subsequentextraction, previously an adsorbate resin based on polystyrene wasalways added, for example Amberlite XAD-1180 (Rohm & Haas, Frankfurt,Germany).

[0013] However, the disadvantage of this process is that, on a largescale, it leads to an abundance of problems. Valves are impaired by theglobules of resin, pipes can block, and apparatus may be subject togreater wear due to mechanical friction. The globules of resin areporous and therefore have a large inner surface area (about 825 m²/gramresin). Sterilisation becomes a problem, as air enclosed in the resin isnot autoclaved. Thus, the process cannot be practicably carried out on alarge scale using resin addition.

[0014] On the other hand, without adding resin globules, a satisfactoryconcentration of epothilones cannot be achieved in the culture medium.

[0015] Surprisingly, the requirements for finding a way out of thisdilemma have now been found, enabling a satisfactory concentration ofnatural substances to be obtained from micro-organisms, in particularmyxobacteria, which produce epothilones such as epothilone A or B, inparticular a concentration of epothilones A and B, in the culturemedium, without the addition of resins, and thus enabling production ofthese compounds, especially epothilones to be carried out on a technicaland industrial scale without the above-mentioned disadvantages.

DETAILED DESCRIPTION OF THE INVENTION

[0016] One aspect of the invention relates to a process forconcentrating epothilones, especially epothilone A and/or B, inparticular epothilone B, in a culture medium, in order to produce thesecompounds on a biotechnological scale, the process comprisingmicroorganisms which produce these compounds, especially myxobacteria(as producers of natural substances), whereby a complex-formingcomponents which is soluble in the culture medium is added to themedium.

[0017] A further aspect relates to the corresponding culture medium,which comprises a corresponding complex-forming component andmicroorganisms, especially myxobacteria, in particular of the genusSorangium, which are suitable for producing epothilones, especiallyepothilone A and/or B.

[0018] A further aspect of the invention relates to a process for theproduction of epothilones, especially epothilone A and/or B, especiallythe two pure compounds, in particular epothilone B, which ischaracterised in that the epothilones are obtained by working up aculture medium for the biotechnological preparation of these compounds,which comprises as producers of natural substances microorganisms,especially myxobacteria, that produce these compounds, and to which acomplex-forming component that is soluble in the culture medium isadded, and the subsequent purification and, if desired, separation ofthe epothilones, for example epothilone A and B.

[0019] A fourth aspect of the invention relates to a method ofseparating epothilones, especially epothilones A and B from one another,which is characterised by chromatography on a reversed-phase column withan eluant comprising a lower alkyl cyanide.

[0020] A further aspect of the invention relates to a strain ofSorangium cellulosum obtained by mutagenesis, which under otherwiseidentical conditions, produces more epothilones than Sorangiumcellulosum Soce90.

[0021] A further aspect also relates to new crystal forms of epothiloneB.

[0022] The general terms used hereinabove and hereinbelow preferablyhave the meanings given hereinbelow:

[0023] Where reference is made hereinabove and hereinbelow to documents,these are incorporated insofar as is necessary.

[0024] The prefix “lower” always indicates that the correspondinglynamed radical contains preferably up to a maximum of 7 carbon atoms, inparticular up to 4 carbon atoms, and is branched or unbranched. Loweralkyl may be for example unbranched or branched once or more, and ise.g. methyl, ethyl, propyl such as isopropyl or n-propyl, butyl such asisobutyl, sec.-butyl, tert.-butyl or n-butyl, or also pentyl such asamyl or n-pentyl.

[0025] A culture medium for the biotechnological preparation ofepothilones which contains micro-organisms that produce these compounds,especially myxobacteria, as producers of natural substances, isprimarily a medium which comprises a corresponding (naturally occurringor also obtainable by cultivation or in particular by geneticmodification) microorganism, especially a myxobacterial strain which isin a position to produce these compounds, in particular a myxobacteriumof the genus Sorangium, preferably (in particular for epothiloneproduction) a microorganism of the type Sorangium Cellulosum Soce90 (seeWO 93/10121), or a biomaterial derived therefrom or from parts of thismyxobacterium, especially a correspondingly derived strain, inparticular the strain having the reference BCE33/10, in particular thestrain having the reference BCE 63/114 or mutants thereof, and inaddition, together with water, preferably other conventional andappropriate constituents of culture media, such as biopolymers, sugar,amino acids, salts, nucleic acids, vitamins, antibiotics,semiochemicals, growth media, extracts from biomaterials such as yeastor other cell extracts, soy meal, starch such as potato starch and/ortrace elements, for example iron ions in complex-bound form, or suitablecombinations of all or some of these constituents and/or also analogousadditions. The corresponding culture media are known to the personskilled in the art or may be produced by known processes (see e.g. theculture media in the examples of the present disclosure, or in WO93/10121).

[0026] One preferred myxobacterium is a strain selected by UVmutagenesis and selection for increased formation of epothilone A and/orB over Sorangium cellulosum Soce90, which is deposited in the DSM underno. 6773, especially the mutant BCE33/10, which was deposited under thenumber DSM 11999 on Feb. 9, 1998 at the German Collection ofMicroorganisms and Cell Cultures (DSMZ, Braunschweig, Germany), and mostpreferably the mutant having the reference BCE 63/114, which wasdeposited under number DSM 12539 on Nov. 27, 1998 at the GermanCollection of Microorganisms and Cell Cultures (DSMZ).

[0027] Strain culture and morphological description of strain BCE 33/10and of strain BCE 63/114: The strain can grow on cellulose as the solesource of carbon and energy with potassium nitrate as the sole source ofnitrogen, e.g. on filter paper over ST21 mineral salt agar (0.1% KNO₃;0.1% MgSO₄x7H₂O; 0.1% CaCl₂x2H₂O; 0.1% K₂HPO₄; 0.01% MnSO₄x7H₂O; 0.02%FeCl₃; 0.002% yeast extract; standard trace element solution; 1% agar).On this medium, dark reddish-brown to blackish-brown fruiting bodies areformed. They consist of small sporangioles (ca. 15 to 30 μm diameter)and exist in dense heaps and packs of varying size.

[0028] The vegetative bacilli have the shape typical of Sorangium(relatively compact, under the phase contrast microscope dark,cylindrical bacilli with broad rounded ends, on average 3 to 6 μm longand 1 μm thick).

[0029] Epothilones are primarily epothilone A and/or B, but also otherepothilones, for example epothilones C and D named in InternationalApplication WO 97/19086 and WO 98/22461, epothilones E and F named in WO98/22461, and further epothilones obtainable from correspondingmicroorganisms.

[0030] A water-soluble complex-forming component is primarily awater-soluble oligo- or poly-peptide derivative or in particular anoligo- or polysaccharide derivative of cyclic or helical structure,which forms an intramolecular cavity, which because of its sufficientlyhydrophobic properties is in a position to bind epothilones, especiallyepothilone A and/or epothilone B. A water-soluble complex-formingcomponent that is especially preferred is one that is selected fromcyclodextrins or (in particular) cyclodextrin derivatives, or mixturesthereof.

[0031] Cyclodextrins are cyclic (α-1,4)-linked oligosaccharides ofα-D-glucopyranose with a relatively hydrophobic central cavity and ahydrophilic external surface area.

[0032] The following are distinguished in particular (the figures inparenthesis give the number of glucose units per molecule):α-cyclodextrin (6), β-cyclodextrin (7), γ-cyclodextrin (8),δ-cyclodextrin (9), ε-cyclodextrin (10), ζ-cyclodextrin (11),η-cyclodextrin (12), and θ-cyclodextrin (13). Especially preferred areδ-cyclodextrin and in particular α-cyclodextrin, β-cyclodextrin orγ-cyclodextrin, or mixtures thereof.

[0033] Cyclodextrin derivatives are primarily derivatives of theabove-mentioned cyclodextrins, especially of α-cyclodextrin,β-cyclodextrin or γ-cyclodextrin, primarily those in which one or moreup to all of the hydroxy groups (3 per glucose radical) are etherifiedor esterified. Ethers are primarily alkyl ethers, especially loweralkyl, such as methyl or ethyl ether, also propyl or butyl ether; thearyl-hydroxyalkyl ethers, such as phenyl-hydroxy-lower-alkyl, especiallyphenyl-hydroxyethyl ether; the hydroxyalkyl ethers, in particularhydroxy-lower-alkyl ethers, especially 2-hydroxyethyl, hydroxypropylsuch as 2-hydroxypropyl or hydroxybutyl such as 2-hydroxybutyl ether;the carboxyalkyl ethers, in particular carboxy-lower-alkyl ethers,especially carboxymethyl or carboxyethyl ether; derivatised carboxyalkylethers, in particular derivatised carboxy-lower-alkyl ether in which thederivatised carboxy is etherified or amidated carboxy (primarilyaminocarbonyl, mono- or di-lower-alkyl-aminocarbonyl, morpholino-,piperidino-, pyrrolidino- or piperazino-carbonyl, or alkyloxycarbonyl),in particular lower alkoxycarbonyl-lower-alkyl ether, for examplemethyloxycarbonylpropyl ether or ethyloxycarbonylpropyl ether; thesulfoalkyl ethers, in particular sulfo-lower-alkyl ethers, especiallysulfobutyl ether; cyclodextrins in which one or more OH groups areetherified with a radical of formula

—O-[alk-O—]_(n—H)

[0034] wherein alk is alkyl, especially lower alkyl, and n is a wholenumber from 2 to 12, especially 2 to 5, in particular 2 or 3;cyclodextrins in which one or more OH groups are etherified with aradical of formula

[0035] wherein R′ is hydrogen, hydroxy, —O-(alk-O)_(z)—H,—O-(alk(-R)—O—)_(p)—H or —O-(alk(-R)—O—)_(q)-alk-CO—Y; alk in all casesis alkyl, especially lower alkyl; m, n, p, q and z are a whole numberfrom 1 to 12, preferably 1 to 5, in particular 1 to 3; and Y is OR₁ orNR₂R₃, wherein R₁, R₂ and R₃ independently of one another, are hydrogenor lower alkyl, or R₂ and R₃ combined together with the linking nitrogensignify morpholino, piperidino, pyrrolidino or piperazine;

[0036] or branched cyclodextrins, in which etherifications or acetalswith other sugars are present, especially glucosyl-,diglucosyl-(G₂-β-cyclodextrin), maltosyl- or dimaltosyl-cyclodextrin, orN-acetylglucosaminyl-, glucosaminyl-, N-acetylgalactosaminyl- orgalactosaminyl-cyclodextrin.

[0037] Esters are primarily alkanoyl esters, in particular loweralkanoyl esters, such as acetyl esters of cyclodextrins.

[0038] It is also possible to have cyclodextrins in which two or moredifferent said ether and ester groups are present at the same time.

[0039] Mixtures of two or more of the said cyclodextrins and/orcyclodextrin derivatives may also exist.

[0040] Preference is given in particular to α-, β- or γ-cyclodextrins orthe lower alkyl ethers thereof, such as methyl-β-cyclodextrin or inparticular 2,6-di-O-methyl-β-cyclodextrin, or in particular the hydroxylower alkyl ethers thereof, such as 2-hydroxypropyl-α-,2-hydroxypropyl-β- or 2-hydroxypropyl-γ-cyclodextrin.

[0041] The cyclodextrins or cyclodextrin derivatives are added to theculture medium preferably in a concentration of 0.02 to 10, preferably0.05 to 5, especially 0.1 to 4, for example 0.1 to 2 percent by weight(w/v).

[0042] Cyclodextrins or cyclodextrin derivatives are known or may beproduced by known processes (see for example U.S. Pat. Nos. 3,459,731;4,383,992; 4,535,152; 4,659,696; EP 0 094 157; EP 0 149 197; EP 0 197571; EP 0 300 526; EP 0 320 032; EP 0 499 322; EP 0 503 710; EP 0 818469; WO 90/12035; WO 91/11200; WO 93/19061; WO 95/08993; WO 96/14090; GB2,189,245; DE 3,118,218; DE 3,317,064 and the references mentionedtherein, which also refer to the synthesis of cyclodextrins orcyclodextrin derivatives, or also: T. Loftsson and M. E. Brewster(1996): Pharmaceutical Applications of Cyclodextrins: DrugSolubilization and Stabilisation: Journal of Pharmaceutical Science 85(10):1017-1025; R. A. Rajewski and V. J. Stella(1996): PharmaceuticalApplications of Cyclodextrins: In Vivo Drug Delivery: Journal ofPharmaceutical Science 85 (11): 1142-1169).

[0043] In the following description of the working up, “epothilone” isunderstood to be an epothilone which is obtainable from thecorresponding microorganism, preferably epothilone C, D, E, F orespecially A or in particular epothilone B. If not otherwise stated,where “epothilones” are mentioned, this is intended to be a general termwhich includes individual epothilones or mixtures.

[0044] Working up of the epothilones is effected by conventionalmethods; first of all, by separating a culture into the liquid phase(centrifugate or filtrate) and solid phase (cells) by means offiltration or centrifugation (tubular centrifuge or separator). The(main) part of the epothilones found in the centrifugate or in thefiltrate is then directly mixed with a synthetic resin, for example aresin based on polystyrene as matrix (hereinafter referred to alsosimply as polystyrene resin), such as Amberlite XAD-16 [Rohm & HaasGermany GmbH, Frankfurt] or Diaion HP-20 [Resindion S. R. L., MitsubishiChemical Co., Milan] (preferably in a ratio of centrifugate: resinvolume of ca. 10:1 to 100:1, preferably about 50:1). After a period ofcontact of preferably 0.25 to 50 hours, especially 0.8 to 22 hours, theresin is separated, for example by filtration or centrifugation. Ifrequired, after adsorption, the resin is washed with a strongly polarsolvent, preferably with water. Desorption of the epothilones is theneffected with an appropriate solvent, especially with an alcohol, inparticular isopropanol. The solvent phase, especially the isopropanolphase, is then removed from the solvent, preferably by means of prior,simultaneous or subsequent addition of water, in particular in acirculating evaporator, thereby being concentrated if necessary, and theresulting water phase is extracted with a solvent suitable for forming asecond phase, such as an ester, for example a lower alkanol loweralkanoate, typically ethyl acetate or isopropyl acetate. The epothilonesare thereby transferred into the organic phase. Then the organic phaseis concentrated to the extent necessary, preferably to dryness, forexample using a rotary evaporator.

[0045] Subsequently, further processing takes place using the followingsteps, whereby the purification step by means of reversed-phasechromatography with elution with a nitrile is an inventive step and isthus compulsory, while the other steps are optional:

[0046] molecular filtration (gel chromatography), e.g. on a column ofmaterial such as Sephadex LH-20 (Pharmacia, Uppsala, Sweden) with analcohol such as methanol as eluant;

[0047] separation of the epothilones by reversed-phase chromatographyafter being taken up in a suitable solvent, and elution with a mixtureof nitrile/water (compulsory), preferably characterised in that thechromatography is carried out on a column of material, especially aRP-18 material, which is charged with hydrocarbon chains, such ashydrocarbon chains containing 18 carbon atoms, and an eluant comprisinga nitrile, especially a lower alkyl-nitrile, in particular acetonitrile,is used, in particular a mixture of nitrile/water is used, especially amixture of acetonitrile/water, preferably in a ratio of nitrile to waterof about 1:99 to 99:1, primarily between 1:9 and 9:1, e.g. between 2:8and 7:3, e.g. 3:7 or 4:6.

[0048] single or multiple extraction of the residue (especially afterevaporation) in a two-phase system consisting of water and a solventimmiscible with water, preferably an ester, in particular a lower alkyllower alkanoate, such as ethyl acetate or isopropyl acetate;

[0049] adsorption chromatography, in particular by adding to a column ofsilica gel and eluting with an appropriate solvent or solvent mixture,especially a mixture of ester/hydrocarbon, for example lower alkylalkanoate/C₄-C₁₀-alkane, especially ethyl or isopropyl acetate/n-hexane,in which the ratio between the ester and hydrocarbon is preferably inthe range 99:1 to 1:99, preferably 10:1 to 1:10, for example 4:1;

[0050] dissolving the residue, which may be obtained afterconcentration, in an appropriate solvent such as an alcohol, e.g.methanol;

[0051] mixing with activated carbon and removal thereof;

[0052] recrystallisation, e.g. from appropriate solvents or solventmixtures, for example consisting of esters, ester/hydrocarbon mixturesor alcohols, especially ethyl or isopropyl acetate:toluene 1:10 to 10:1,preferably 2:3 (epothilone A) or methanol or ethyl acetate (epothiloneB);

[0053] whereby between each step being employed, the resulting solutionsor suspensions are concentrated if necessary, and/or liquid and solidcomponents are separated from one another, in particular by filtering orcentrifuging solutions/suspensions. The more precise definitionsmentioned below can be preferably used in the above individual steps.

[0054] The further working up and purification is preferably carried out

[0055] either by direct separation of the epothilones from one anotherby reversed-phase chromatography after being taken up in an appropriatesolvent, for example a nitrile/water mixture, especially anacetonitrile/water mixture (ratio of nitrile to water 1:99 to 99:1,preferably 1:9 to 9:1, especially 3:1), if necessary after filtration orcentrifugation, preferably on a silica gel that has been derivatized byhydrocarbon radicals, e.g. a silica gel modified by alkyl radicalscontaining 8 to 20, especially 18, C-atoms, eluting with an eluantcomprising a nitrile, especially a lower alkylnitrile, such asacetonitrile, especially a mixture of the nitrile with water, such as anacetonitrile/water mixture, whereby detection of the interestingfractions is effected in conventional manner, for example by UVdetection or (preferably) by on-line HPLC (HPLC with a very smallcolumn, the analyses taking less than 1 minute, and detection e.g. at250 nm), this enabling a particularly exact separation of the fractionscontaining the desired product to take place; if required, withsubsequent concentration, for example by distillation, to remove thenitrile; if desired, with subsequent single or multiple, for exampledouble, extraction of the residue of evaporation in a two-phase systemconsisting of water and an immiscible solvent, such as ethyl acetate orisopropyl acetate; additional concentration of the organic phase anddissolving of the residue in an appropriate solvent, preferably an estersuch as ethyl acetate or isopropyl acetate, if required, filtration orcentrifugation, if desired adding to a column of silica gel and elutingwith an appropriate solvent or solvent mixture, for example with amixture of ester/hydrocarbon, e.g. lower alkyl alkanoate/C₄-C₁₀-alkane,especially ethyl or isopropyl acetate/n-hexane, in which the ratio ofester to hydrocarbon is preferably in the range 99:1 to 1:99, preferably10:1 to 1:10, e.g. 4:1; subsequent combining of the fractions containingeach desired epothilone, especially epothilone A or epothilone B, andafter removing the solvent, for example by distillation, preferablyconcentrating to dryness; then, dissolving of the residue in anappropriate alcohol, preferably methanol; and if desired, in order toobtain especially high purity, mixing with activated carbon and thenseparating the activated carbon, for example by filtration; and finally,by recrystallisation as described below under variant 2 (for epothiloneB in particular from methanol), separate extraction of the epothilones,especially epothilones A or B. This is the most preferred variant 1, theoutstanding characteristic of which is the surprising direct separationby reversed-phase chromatography of the epothilone-containing mixturedesorbed by the resin, despite all the impurities in the organicextract;

[0056] or (variant 2) first of all exclusion chromatography takes place(molecular filtration) e.g. on a column of material such as SephadexLH-20 (Pharmacia, Uppsala, Sweden) with an alcohol such as methanol aseluant, and then subsequent separation of the epothilones present in thepeak fractions obtained, e.g. epothilone A and B, by reversed-phasechromatography as described above for variant 1; if required twice, ifpeak fractions of one epothilone contain those of another, for example1f those with epothilone A still contain residues of epothilone B; andthen separate recrystallisation of each epothilone from appropriatesolvents or solvent mixtures, for example from ethyl or isopropylacetate:toluene 1:10 to 10:1, preferably 2:3 (epothilone A) or methanolor ethyl acetate (epothilone B). This is variant 2 of working up andpurification.

[0057] With variant 1, highly pure epothilone B may be obtained in arelatively simpler manner (the purity is preferably greater than 97%,especially over 99%).

[0058] Variant 1 preferably takes place as follows (whereby preferablythe above-mentioned variants can be used instead of the followinggeneral definitions): First of all, a culture is separated into theliquid phase (centrifugate or filtrate) and a solid phase (cells) bymeans of filtration or centrifugation (tubular centrifuge or separator).The (main) part of the epothilones found in the centrifugate or filtrateis then directly mixed with a synthetic resin. After a contact period ofpreferably 0.25 to 50 hours, the resin is separated, for example byfiltration or centrifugation. If required, after adsorption, the resinis washed with a strongly polar solvent, preferably with water.Desorption of the epothilones is then effected with an appropriatesolvent, especially with an alcohol, in particular isopropanol. Thesolvent phase, especially isopropanol phase, is then removed from thesolvent, preferably by means of prior, simultaneous or subsequentaddition of water, in particular in a circulating evaporator, therebybeing concentrated if necessary, and the resulting water phase isextracted with a solvent suitable for forming a second phase, such as anester, for example a lower alkanol lower alkanoate, typically ethylacetate or isopropyl acetate. The epothilones are then transferred intothe organic phase. Then the organic phase is concentrated to the extentnecessary, preferably to dryness, for example using a rotary evaporator.By subsequent reversed-phase chromatography on a silica gel derivatizedwith hydrocarbon atoms, e.g. a silica gel modified by alkyl radicalscontaining 18 C-atoms, and eluting with a mixture of a loweralkylnitrile such as acetonitrile with water, the epothilones aredirectly separated from one another, especially epothilone A andepothilone B; then, concentration takes place by means of distillation,the residue is shaken out once or more, if desired from water with anappropriate solvent that is immiscible with water, preferably an estersuch as isopropyl acetate, then the organic phase is again concentratedand the residue of evaporation is dissolved in an ester such as ethyl orisopropyl acetate, filtered if required, the filtrate added to a columnof silica gel, and eluted with a mixture of ester/hydrocarbon, e.g.ethyl or isopropyl acetate/n-hexane; subsequently, the fractionscontaining the epothilone, especially epothilone A or B, arerespectively combined and, after removing the solvent by distillation,concentrated, preferably to dryness; the residue is then dissolved in anappropriate lower alkanol, preferably methanol, and in order to obtainespecially high purity, mixed with activated carbon and then filtered;finally, the epothilones are extracted by recrystallisation (in the caseof epothilone B preferably from methanol).

[0059] Variant 2 is effected preferably as follows: After harvest, aculture is separated into the liquid phase (centrifugate) and solidphase (cells) by means of centrifugation (tubular centrifuge orseparator). The main part of the epothilones are found in thecentrifugate, which is then directly mixed with a polystyrene resin,such as Amberlite XAD-16 [Rohm & Haas Germany GmbH, Frankfurt] or DiaionHP-20 [Resindion S. R. L., Mitsubishi Chemical Co., Milan] (preferablyin a ratio of centrifugate: resin volume of ca. 10:1 to 100:1,preferably about 50:1) and stirred in an agitator. In this step, theepothilones are transferred from the cyclodextrin to the resin. After aperiod of contact of ca. 1 hour, the resin is separated bycentrifugation or filtration. Adsorption of the epothilones onto theresin may also be effected in a chromatography column, by placing theresin in the column and running the centrifugate over the resin. Afteradsorption, the resin is washed with water. Desorption of theepothilones from the resin is effected with isopropanol. The isopropanolphase is then freed of isopropanol preferably by the addition of waterin particular in a circulating evaporator, and the resulting water phaseis extracted with ethyl acetate. The epothilones are thus transferredfrom the water phase to the ethyl acetate phase. Then the ethyl acetateextract is concentrated to dryness, for example using a rotaryevaporator. An initial concentration of the epothilones is then achievedby means of molecular filtration (e.g. Sephadex LH-20 [Pharmacia,Uppsala, Sweden] with methanol as eluant). The peak fractions from themolecular filtration contain epothilone A and B and have a totalepothilone content of >10%. Separation of these peak fractions, whichcontain epothilone A and B in a mixture, into the individual components,then follows by means of chromatography on a “reversed-phase”, e.g.RP-18 phase (phase which is modified by alkyl radicals containing 18carbon atoms per chain), with an appropriate eluant, preferably onecontaining a nitrile such as acetonitrile (this gives better separationthan for example alcohols such as methanol). Epothilone A elutes beforeepothilone B. The peak fractions with epothilone B may still containsmall portions of epothilone A, which can be removed by furtherseparation on RP-18. Finally, the epothilone A fraction is crystalliseddirectly from ethyl acetate:toluene=2:3, and the epothilone B fractionfrom methanol or ethyl acetate.

PREFERRED EMBODIMENT OF THE INVENTION

[0060] The invention preferably relates to a process for theconcentration of epothilones, especially epothilone A and/or B, inparticular epothilone B, in a culture medium for the biotechnologicalpreparation of this (these) compound(s), which contains a microorganismwhich is suitable for this preparation, especially a Sorangium strain,especially of the type Sorangium Cellulosum Soce90, or a mutant arisingtherefrom, in particular the strain having reference BCE 33/10,especially the strain having reference BCE 63/114, water and other usualappropriate constituents of culture media, whereby a cyclodextrin or acyclodextrin derivative, or a mixture of two or more of these compoundsis added to the medium, especially one or more, preferably one or two ormore cyclodextrins selected from α-cyclodextrin (6), β-cyclodextrin (7),γ-cyclodextrin (8), δ-cyclodextrin (9), ε-cyclodextrin (10),ζ-cyclodextrin (11), η-cyclodextrin (12), and θ-cyclodextrin (13),especially α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin; orprimarily a cyclodextrin derivative or mixture of cyclodextrinderivatives selected from derivatives of a cyclodextrin, in which one ormore up to all of the hydroxy groups are etherified to an alkyl ether,especially lower alkyl, such as methyl or ethyl ether, also propyl orbutyl ether; an aryl-hydroxyalkyl ether, such asphenyl-hydroxy-lower-alkyl, especially phenyl-hydroxyethyl ether; ahydroxyalkyl ether, in particular hydroxy-lower-alkyl ethers, especially2-hydroxyethyl, hydroxypropyl such as 2-hydroxypropyl or hydroxybutylsuch as 2-hydroxybutyl ether; a carboxyalkyl ether, in particularcarboxy-lower-alkyl ether, especially carboxymethyl or carboxyethylether; a derivatised carboxyalkyl ether, in particular a derivatisedcarboxy-lower-alkyl ether in which the derivatised carboxy isaminocarbonyl, mono- or di-lower-alkyl-aminocarbonyl, morpholino-,piperidino-, pyrrolidino- or piperazino-carbonyl, or alkyloxycarbonyl,in particular lower alkoxycarbonyl, such as preferably loweralkoxycarbonyl-lower-alkyl ether, for example methyloxycarbonylpropylether or ethyloxycarbonylpropyl ether; a sulfoalkyl ether, in particularsulfo-lower-alkyl ether, especially sulfobutyl ether; a cyclodextrin inwhich one or more OH groups are etherified with a radical of formula

—O-[alk-O—]_(n)—H

[0061] wherein alk is alkyl, especially lower alkyl, and n is a wholenumber from 2 to 12, especially 2 to 5, in particular 2 or 3; acyclodextrin in which one or more OH groups are etherified with aradical of formula

[0062] wherein R′ is hydrogen, hydroxy, —O-(alk-O)_(n)—H,—O-(Alk(-R)—O—)_(p)—H or —O-(alk(-R)—O—)_(q)-alk-CO—Y; alk in all casesis alkyl, especially lower alkyl; m, n, p, q and z are a whole numberfrom 1 to 12, preferably 1 to 5, in particular 1 to 3; and Y is OR₁ orNR₂R₃, wherein R₁, R₂ and R₃ independently of one another, are hydrogenor lower alkyl, or R₂ and R₃ combined together with the linking nitrogensignify morpholino, piperidino, pyrrolidino or piperazino; or a branchedcyclodextrin, in which etherifications or acetals with other sugars arepresent, and which are selected from glucosyl-,diglucosyl-(G₂-β-cyclodextrin), maltosyl- or dimaltosyl-cyclodextrin, orN-acetylglucosaminyl-, glucosaminyl-, N-acetylgalactosaminyl- andgalactosaminyl-cyclodextrin; or a lower alkanoyl, such as acetyl esterof a cyclodextrin.

[0063] Particular preference is given to a process in which thecyclodextrin and/or the cyclodextrin derivative is added to the culturemedium in a concentration of 0.02 to 10, preferably 0.005 to 10, morepreferably 0.05 to 5, most preferably 0.1 to 4, for example 0.1 to 2,percent by weight (w/v).

[0064] Especially preferred is a process according to one of the twoprevious paragraphs, in which the cyclodextrin derivative is selectedfrom a cyclodextrin, especially β-cyclodextrin, and a hydroxy loweralkyl-cyclodextrin, especially 2-hydroxypropyl-α-, -β- or-γ-cyclodextrin; or mixtures of one or more thereof; whereby2-hydroxypropyl-β-cyclodextrin on its own is preferred in particular.

[0065] The invention also relates in particular to a culture medium,which comprises a cyclodextrin, a cyclodextrin derivative or a mixtureof two or more complex-forming components selected from cyclodextrinsand cyclodextrin derivatives, especially a cyclodextrin or cyclodextrinderivative as defined in the third-last paragraph, in particular as inthe second-last paragraph, or a mixture of one or more of thesecompounds, and a microorganism which is suitable for producingepothilones, especially epothilone A and/or B, preferably a strain fromthe genus Sorangium, especially a strain of Sorangium Cellulosum, e.g.the strain Soce90 or a mutant arising therefrom, in particular thestrain BCE 33/10, or especially BCE 63/114.

[0066] A further aspect of the invention relates to a process for theproduction of epothilone A and/or B, especially the two pure compounds,in particular epothilone B, which is characterised in that theepothilones are separated for example by centrifugation into the solidand the liquid phase (centrifugate) by working up a culture medium forthe biotechnological preparation of these compounds, as described above,to which has been added a complex-forming component which is soluble inthe culture medium, in particular a cyclodextrin, a cyclodextrinderivative or a mixture of two or more cyclodextrins and/or cyclodextrinderivatives; the centrifugate is mixed with a resin, especially apolystyrene resin, or is run through a column filled with such a resin;if necessary, the resin is washed with water; the epothilone(s) is orare desorbed from the resin using a polar solvent, especially analcohol, primarily a lower alkanol such as isopropanol; if necessary,concentrated by means of prior, simultaneous or subsequent addition ofwater; an organic solvent which is immiscible with water, for example anester, such as ethyl acetate, is added, and the epothilone(s) is or aretransferred to the organic phase, for example by agitating or stirring;where necessary, the organic phase is concentrated; the epothilones fromthe organic solution obtained are concentrated through a molecular sievefor compounds of low molecular weight; and then the fractions containingthe epothilones, especially epothilone A and/or B undergo separation bya reversed-phase column, preferably eluting with an eluant containing anitrile, such as acetonitrile (or instead, an eluant containing analcohol, such as methanol); whereby epothilones A and B are extractedseparately, and if desired, can be further concentrated byrecrystallisation.

[0067] One preferred aspect of the invention relates to a process forthe production of epothilone A and/or B, especially the two purecompounds, in particular epothilone B, which is characterised in thatthe epothilones are separated for example by centrifugation into thesolid and the liquid phase (centrifugate) by working up a culture mediumfor the biotechnological preparation of these compounds, as describedabove, to which has been added a complex-forming component which issoluble in the culture medium, in particular a cyclodextrin, acyclodextrin derivative or a mixture of two or more cyclodextrins and/orcyclodextrin derivatives; the centrifugate is mixed with a resin,especially a polystyrene resin, or is run through a column filled withsuch a resin; if necessary, the resin is washed with water; theepothilone(s) is or are desorbed from the resin using a polar solvent,especially an alcohol, primarily a lower alkanol such as isopropanol; ifnecessary, the polar solvent is removed by means of prior, simultaneousor subsequent addition of water; the resulting water phase is extractedwith a solvent which is suitable for forming a second phase, for examplean ester, such as diethyl ester, if necessary, the organic phase isconcentrated, preferably to dryness; epothilone A and B are separatedfrom one another directly by reversed-phase chromatography, eluting withan eluant containing a nitrile, especially a lower alkylnitrile, such asacetonitrile, whereby detection is effected in the usual manner, forexample by UV detection or preferably by on-line HPLC (HPLC with a verysmall column, the analyses taking less than 1 minute, and detection e.g.at 250 nm); subsequent concentration, for example by distillation; ifdesired, the residue is treated from an aqueous solution once or more(for example twice) by extraction with a solvent which is immisciblewith water, such as an ester; dissolved in an appropriate solvent,preferably an ester such as ethyl or isopropyl acetate, filtered ifnecessary, added to a column of silica gel and eluted with anappropriate solvent or solvent mixture, for example with anester/hydrocarbon mixture; and subsequently, the fractions containingeither epothilone A or especially B are separately combined and, afterremoving the solvent, for example by distillation, concentratedpreferably to dryness; then the residue is dissolved in an appropriatealcohol, preferably methanol, then if desired, in order to obtainespecially high purity, treated with activated carbon and then filtered;and finally epothilone A or B is obtained by recrystallisation (in thecase of epothilone B, particularly from methanol).

[0068] A further preferred aspect of the invention relates to a methodof separating epothilones, especially epothilones A and B from oneanother, which is characterised by chromatography on a reversed-phasecolumn with an eluant containing a lower alkyl cyanide, chromatographybeing carried out on a column material, especially an RP-18 material,which is charged with hydrocarbon chains containing 18 carbon atoms, andemploying an eluant containing a nitrile, especially a loweralkylnitrile, in particular acetonitrile, especially a mixture ofnitrile/water, in particular a mixture of acetonitrile/water, preferablyin a ratio of nitrile to water of ca. 1:99 to 99:1, primarily 1:9 to9:1, e.g. between 2:8 and 7:3, e.g. 3:7 or 4:6. This separation mayfollow on to a filtration with a molecular sieve, or is preferablyeffected directly using the residue after adsorption of the epothilonesfrom the culture medium containing a complex-forming component onto aresin, as described above (“variant 1”). One advantage of separationwith an eluant containing a lower alkylcyanide over that using alcohols,such as methanol, is the better separation of epothilones A and B.

[0069] The invention relates preferably to a process for the preparationof epothilones, which a) comprises a process for the concentration ofepothilones, especially epothilone A and/or B, in particular epothiloneB, in a culture medium for the biotechnological preparation of this(these) compound(s), which contains a microorganism which is suitablefor this preparation, especially a Sorangium strain, especially of thetype Sorangium Cellulosum Soce90, or a mutant arising therefrom, inparticular the strain having reference BCE 33/10, especially the strainhaving reference BCE 63/114, water and other usual appropriateconstituents of culture media, whereby a cyclodextrin or a cyclodextrinderivative, or a mixture of two or more of these compounds is added tothe medium, especially one or more, preferably one or two or morecyclodextrins selected from α-cyclodextrin (6), β-cyclodextrin (7),γ-cyclodextrin (8), δ-cyclodextrin (9), ε-cyclodextrin (10),ζ-cyclodextrin (11), η-cyclodextrin (12), and θ-cyclodextrin (13),especially α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin; orprimarily a cyclodextrin derivative or mixture of cyclodextrinderivatives selected from derivatives of a cyclodextrin, in which one ormore up to all of the hydroxy groups are etherified to an alkyl ether,especially lower alkyl, such as methyl or ethyl ether, also propyl orbutyl ether; an aryl-hydroxyalkyl ether, such asphenyl-hydroxy-lower-alkyl, especially phenyl-hydroxyethyl ether; ahydroxyalkyl ether, in particular hydroxy-lower-alkyl ether, especially2-hydroxyethyl, hydroxypropyl such as 2-hydroxypropyl or hydroxybutylsuch as 2-hydroxybutyl ether; a carboxyalkyl ether, in particularcarboxy-lower-alkyl ether, especially carboxymethyl or carboxyethylether; a derivatised carboxyalkyl ether, in particular a derivatisedcarboxy-lower-alkyl ether in which the derivatised carboxy isaminocarbonyl, mono- or di-lower-alkyl-aminocarbonyl, morpholino-,piperidino-, pyrrolidino- or piperazinocarbonyl, or alkyloxycarbonyl, inparticular lower alkoxycarbonyl, such as preferably a loweralkoxycarbonyl-lower-alkyl ether, for example methyloxycarbonylpropylether or ethyloxycarbonylpropyl ether; a sulfoalkyl ether, in particularsulfo-lower-alkyl ether, especially sulfobutyl ether; a cyclodextrin inwhich one or more OH groups are etherified with a radical of formula

—O-[alk-O—]_(n)—H

[0070] wherein alk is alkyl, especially lower alkyl, and n is a wholenumber from 2 to 12, especially 2 to 5, in particular 2 or 3; acyclodextrin in which one or more OH groups are etherified with aradical of formula

[0071] wherein R′ is hydrogen, hydroxy, —O-(alk-O)_(n)—H,—O-(Alk(-R)-O-)_(p)-H or —O-(alk(-R)—O—)_(q)-alk-CO—Y; alk in all casesis alkyl, especially lower alkyl; m, n, p, q and z are a whole numberfrom 1 to 12, preferably 1 to 5, in particular 1 to 3; and Y is OR₁ orNR₂R₃, wherein R₁, R₂ and R₃ independently of one another, are hydrogenor lower alkyl, or R₂ and R₃ combined together with the linking nitrogensignify morpholino, piperidino, pyrrolidino or piperazino;

[0072] or a branched cyclodextrin, in which etherifications or acetalswith other sugars are present, and which are selected from glucosyl-,diglucosyl-(G₂-β-cyclodextrin), maltosyl- or di-maltosyl-cyclodextrin,or N-acetylglucosaminyl-, glucosaminyl-, N-acetylgalactosaminyl- andgalactosaminyl-cyclodextrin; or a lower alkanoyl, such as acetyl esterof a cyclodextrin; and

[0073] b) comprises a step for separating the epothilones, especiallyepothilones A and B, from one another, which is characterised bychromatography on a reversed-phase column with an eluant containing alower alkylcyanide, the chromatography being carried out on a columnmaterial, especially an RP-18 material, which is charged withhydrocarbon chains containing 18 carbon atoms, and employing an eluantcontaining a lower alkylnitrile, especially acetonitrile, in particulara mixture of lower alkylnitrile/water, preferably a mixture ofacetonitrile/water, preferably in a ratio of lower alkylnitrile to waterof ca. 1:99 to 99:1, primarily 1:9 to 9:1, e.g. between 2:8 and 7:3,e.g. 3:7 or 4:6, whereby if desired, it is possible to use further stepsfor working up and purification.

[0074] The invention also relates in particular to a mutant derived fromthe strain Sorangium cellulosum Soce90, especially a strain of Sorangiumcellulosum which is obtainable by mutagenesis, preferably by one or moreUV-induced mutagenesis steps (in particular by UV radiation in the range200 to 400, especially 250 to 300 nm) with subsequent searching in eachstep for mutants having increased epothilone production (in particularincreased epothilone concentration in the culture medium), this strainunder otherwise identical conditions producing more epothilones, inparticular more epothilone A and/or B, especially epothilone B, thanSorangium cellulosum Soce90, especially the Sorangium cellulosum strainBCE 33/10, in particular BCE 114.

[0075] The invention relates in particular to the individual processsteps named in the examples or any combination thereof, the culturemedia named therein, crystal forms and the strain described therein.

[0076] The invention also relates to new crystal forms of epothilone B,especially a crystal form of epothilone B described as modification Band in particular described as modification A.

[0077] The crystal forms can be distinguished in particular by theirX-ray diagrams. X-ray diagrams taken with a diffractometer and usingCu-Kα₁-radiation are preferably used to characterize solids of organiccompounds. X-ray diffraction diagrams are used particularly successfullyto determine the crystal modification of a substance. To characterizethe existing crystal modification A and crystal modification B ofepothilone B, the measurements are made at an angle range (20) of 2° and35° with samples of substance that are kept at room temperature.

[0078] The X-ray diffraction diagram thus determined (reflection linesand intensities of the most important lines) from crystal modification A(modification A) of epothilone B is characterized by the followingtable. 2θ Intensity 7.7 very strong 10.6 weak 13.6 average 14.4 average15.5 average 16.4 weak 16.8 weak 17.1 weak 17.3 weak 17.7 weak 18.5 weak20.7 strong 21.2 strong 21.9 weak 22.4 weak 23.3 strong 25.9 average31.2 weak 32.0 average

[0079] The invention also relates in particular to a new crystal form ofepothilones B, which is characterised by a melting point of more than120° C., especially between 120° C. and 128° C., in particular 124-125°C. Surprisingly, this value is considerably higher than the valuespreviously described in litreature. The invention relates especially toa crystal form of epothilone B, which is characterised by the X-raydiffraction diagram of the crystal form A and a melting point of above120° C., especially between 120° C. and 128° C., for example between124° C. and 125° C.

[0080] The X-ray diffraction diagram thus determined (reflection linesand intensities of the most important lines) of crystal modification B(modification B) of epothilone B is characterized by the followingtable. 2θ Intensity 6.9 very strong 8.0 weak 8.3 average 10.8 strong11.5 average 12.4 weak 13.1 strong 15.5 weak 16.2 weak 16.7 average 18.1average 18.6 average 20.4 weak 20.9 strong 21.3 weak 21.5 very weak 22.5average 24.2 weak 25.1 average

[0081] The invention also relates in particular to a new crystal form ofepothilones B, which is characterised by a melting point of more than120° C., especially between 124 and 125° C. Surprisingly, this value isconsiderably higher than the values previously described in litreature.

[0082] The new crystal forms are especially stable, particularly crystalform A, and they are therefore suitable as active ingredients for solidforms of administration, for storing in solid form or as intermediates(with particularly good storability) in the preparation of solid orliquid forms of administration.

[0083] The invention also relates to the use of the new crystal forms,especially crystal form B, but primarily crystal form A (all referred tohereinafter as active ingredient) in the production of pharmaceuticalpreparations, new pharmaceutical preparations which contain these newcrystal forms, and/or the use of these compounds in the treatment ofproliferative diseases, such as tumours. In the following, wherepharmaceutical preparations or compositions which comprise or containthe active ingredient are mentioned, in the case of liquid compositionsor compositions which no longer contain the crystal form as such, thisis always understood to mean also the pharmaceutical preparationsobtainable using the crystal forms (for example infusion solutionsobtained using crystal forms A or B of epothilone B), even if they nolonger contain the respective crystal form (for example because theyexist in solution).

[0084] The invention also relates especially to the use of a new crystalform of epothilone B, especially the crystal form B or in particularcrystal form A, in the production of pharmaceutical preparations,characterised by mixing a new crystal form of epothilone B with one ormore carriers.

[0085] The invention also relates to a method of treating warm-bloodedanimals suffering from a proliferative disease, characterised byadministering a dose of epothilone B which is effective for treatingsaid disease in one or the new crystal forms to a warm-blooded animalrequiring such treatment, also including in particular the treatmentwith those preparations that are produced using one of the new crystalforms.

[0086] To produce the pharmaceutical preparations, the active ingredientmay be used for example in such a way that the pharmaceuticalpreparations contain an effective amount of the active ingredienttogether or in a mixture with a significant amount of one or moreorganic or inorganic, liquid or solid, pharmaceutically acceptablecarriers.

[0087] The invention also relates to a pharmaceutical composition whichis suitable for administration to a warm-blooded animal, especiallyhumans, in the treatment of a proliferative disease, such as a tumour,the composition containing an amount of active ingredient that issuitable for treating said disease, together with a pharmaceuticallyacceptable carrier.

[0088] The pharmaceutical compositions according to the invention arethose intended for enteral, especially nasal, rectal or oral, orpreferably parenteral, especially intramuscular or intravenousadministration to warm-blooded animals, especially humans, and theycontain an effective dose of the active ingredient on its own ortogether with a significant amount of a pharmaceutically acceptablecarrier. The dose of the active ingredient is dependent on the type ofwarm-blooded animal, the body weight, the age and the individualcondition, individual pharmacokinetic situations, the disease to betreated and the type of administration.

[0089] The pharmaceutical compositions contain ca. 0.0001% to ca. 95%,preferably 0.001% to 10% or 20% to ca. 90% of active ingredient.Pharmaceutical compositions according to the invention may be presentfor example in unit dose forms, such as in the form of ampoules, vials,suppositories, dragees, tablets or capsules.

[0090] The pharmaceutical compositions according to the presentinvention are produced by known processes, for example by conventionaldissolving, lyophilizing, mixing, granulating or manufacturingprocesses.

[0091] Solutions of the active ingredient, also suspensions, and inparticular aqueous solutions or suspensions, are preferably employed,whereby it is also possible, for example in the case of lyophilisedcompositions which contain the active ingredient on its own or togetherwith a pharmaceutically acceptable carrier, for example mannitol, forthe solutions or suspensions to be prepared prior to administration. Thepharmaceutical compositions may be sterilised and/or may containexcipients, for example preservatives, stabilisers, moisture-retainingagents and/or emulsion-forming agents, dissolving aids, salts forregulating osmotic pressure and/or buffers, and they are produced byknown processes, for example by conventional dissolving or lyophilisingprocesses. The solutions or suspensions mentioned may compriseviscosity-increasing substances, such as sodium carboxymethylcellulose,carboxymethylcellulose, dextran, polyvinylpyrrolidone or gelatin.

[0092] Suspensions in oil contain as the oil component vegetable oils,synthetic oils or semi-synthetic oils, which are customary for injectionpurposes. Notable examples are in particular liquid fatty acid esters,which contain as the acid component a long-chained fatty acid with 8 to22, especially 12 to 22, carbon atoms, for example lauric acid,tridecylic acid, myristic acid, pentadecylic acid, palmitic acid,margaric acid, stearic acid, arachidic acid, behenic acid orcorresponding unsaturated acids, for example oleic acid, elaidic acid,erucic acid, brassidic acid or linoleic acid, if desired with theaddition of antioxidants, for example vitamin E, β-carotene or3,5-di-tert-butyl-4-hydroxytoluene. The alcoholic component of thesefatty acid esters preferably has a maximum of 6 carbon atoms and is amono- or polyhydroxy alcohol, for example a mono-, di- or tri-hydroxyalcohol, for example methanol, ethanol, propanol, butanol or pentanol,or an isomer thereof, but especially glycol and glycerol. The followingexamples of fatty acid esters may be mentioned in particular: propylmyristate, isopropyl palmitate, “Labrafil M 2375” (polyoxyethyleneglycerol trioleate, Gattefosse, Paris), “Miglyol 812” (triglyceride ofsaturated fatty acids having a chain length of 8 to 12 carbon atoms,Hüls AG, Germany), but in particular vegetable oils such as cottonseedoil, almond oil, olive oil, castor oil, sesame oil, soybean oil and inparticular peanut oil.

[0093] The injection or infusion preparations are produced according tocustomary methods under sterile conditions; the same applies also to thefilling of the compositions into ampoules or vials and sealedcontainers.

[0094] Preference is given to an infusion solution which contains theactive ingredient and a pharmaceutically acceptable organic solvent.

[0095] The pharmaceutically acceptable organic solvents which may beused in a formulation according to the invention can be selected fromall such solvents which are familiar to a person skilled in the art. Thesolvent is preferably selected from an alcohol, e.g. absolute ethanol,ethanol/water mixtures, preferably 70% ethanol, polyethylene glycol 300,polyethylene glycol 400, polypropylene glycol and N-methylpyrrolidone,especially polypropylene glycol or 70% ethanol.

[0096] Particular preference is given to a formulation in purepolyethylene glycol, which is diluted prior to infusion in anappropriate solution, such as physiological saline.

[0097] The active ingredient is present in the formulation in aconcentration of 0.001 to 100 mg/ml, preferably from ca. 0.05 to 5mg/ml, or from 5 to 50 mg/ml.

[0098] Formulations of this type are easily stored as vials or ampoules.The vials or ampoules are typically made of glass, e.g. boron silicate.The vials or ampoules may be appropriate for any volume which is knownfrom the prior art. They are preferably of sufficient size to be able toaccept 0.5 to 5 ml of the formulation.

[0099] Prior to administration, the formulations have to be diluted inan aqueous medium suitable for intravenous administration before theactive ingredient can be administered to patients.

[0100] It is preferable for the infusion solution to have the same orbasically the same osmotic pressure as body fluids. Consequently, theaqueous medium contains an isotonic agent which has the effect ofrendering the osmotic pressure of the infusion solution the same orbasically the same as the osmotic pressure of body fluids.

[0101] The isotonic agent can be selected from all agents that arefamiliar to a person skilled in the art, for example mannitol, dextrose,glucose and sodium chloride. The isotonic agent is preferably glucose orsodium chloride. The isotonic agents may be used in quantities whichimpart the same or basically the same osmotic pressure to the infusionsolution as body fluids. The exact quantities required can be determinedby routine experiments and depend on the composition of the infusionsolution and the type of isotonic agent.

[0102] The concentration of isotonizing agent in the aqueous mediumdepends on the type of each agent used. If glucose is used, it ispreferably used in a concentration of 1 to 5% w/v, preferably 5% w/v. Ifthe isotonizing agent is sodium chloride, it is preferably used inquantities of up to 1%, preferably ca. 0.9% w/v.

[0103] The infusion solution can be diluted with the aqueous medium. Theamount of aqueous medium used is chosen according to the desiredconcentration of active ingredient in the infusion solution. Theinfusion solution is preferably produced by mixing a vial or an ampoulecontaining the infusion concentrate (see above) with an aqueous medium,so that a volume of between 200 ml and 1000 ml is attained with theaqueous medium. Infusion solutions may contain other additives that arenormally used in formulations for intravenous administration. Theseadditives also include antioxidants.

[0104] Antioxidants may be used to protect the active ingredient fromdegradation by oxidation. Antioxidants may be selected from those whichare familiar to the person skilled in the art and which are suitable forintravenous formulations. The amount of antioxidant can be determined byroutine experiments. As an alternative to adding an antioxidant, oradditionally thereto, the antioxidant effect can be achieved byrestricting the oxygen (air) contact with the infusion solution. Thiscan be achieved in a simple way, by treating the vessel containing theinfusion solution with an inert gas, e.g. nitrogen or argon.

[0105] Infusion solutions can be produced by mixing an ampoule or a vialwith the aqueous medium, e.g. a 5% glucose solution in WFI in anappropriate container, e.g. an infusion bag or an infusion bottle.

[0106] Containers for the infusion solutions may be selected fromconventional containers that are non-reactive with the infusionsolution. Among those suitable are glass containers, especially of boronsilicate, but plastic containers such as plastic infusion bags, arepreferred.

[0107] Plastic containers may also be made of thermoplastic polymers.The plastic materials may also contain additives, e.g. softeners,fillers, antioxidants, antistatic agents or other customary additives.

[0108] Suitable plastics for the present invention should be resistantto elevated temperatures used for sterilisation. Preferred plasticinfusion bags are the PVC materials which are known to the personskilled in the art.

[0109] A large range of container sizes may be considered. Whenselecting the size of the container, the factors to be taken intoconsideration are especially the solubility of epothilones in an aqueousmedium, easy handling, and if appropriate, storage of the container. Itis preferable to use containers which hold between ca. 200 and 1000 mlof infusion solution.

[0110] Owing to their good formulating properties, the new crystal formsof epothilone B according to the invention are especially suitable forthe simple and reproducible production of the said infusion solutions.However, the new crystal forms are especially suitable for theproduction of pharmaceutical formulations which contain the activeingredient in solid form, for example oral formulations.

[0111] Pharmaceutical formulations for oral application may be obtainedby combining the active ingredient with solid carriers, if desired bygranulating the resultant mixture, and further processing the mixture,if desired or if necessary, after adding suitable adjuvants, intotablets, dragee cores or capsules. It is also possible to embed them inplastic substrates which enable the active ingredient to be diffused orreleased in measured quantities.

[0112] Suitable pharmaceutically employable carriers are especiallyfillers, such as lactose, saccharose, mannitol or sorbitol, cellulosepreparations, and/or calcium phosphates, for example tricalciumphosphate or calcium hydrogen phosphate, and binders, such as starches,for example maize, wheat, rice or potato starch, gelatin, tragacanth,methyl cellulose, hydroxypropyl methyl cellulose, sodiumcarboxymethylcellulose, and/or polyvinyl pyrrolidone, and/or, ifdesired, disintegrators, such as the above-mentioned starches,crosslinked vinylpyrrolidones, agar, alginic acid or a salt thereof,such as sodium alginate. Adjuvants are in particular flow-improvingagents and lubricants, e.g. silicates, talcum, stearic acid or saltsthereof, such as magnesium or calcium stearate and/or polyethyleneglycol. Dragee cores are provided, if desired, with appropriategastricjuice-resistant coatings, using inter alia concentrated sugarsolutions, gum arabic, talcum, polyvinyl pyrrolidone, polyethyleneglycol and/or titanium dioxide, or coating solutions in suitable organicsolvents, or in order to produce gastric-juice-resistant coatings,solutions of appropriate cellulose preparations, such as ethyl cellulosephthalate or hydroxypropyl methyl cellulose phthalate. Capsules are drycapsules consisting of gelatin or pectin, and if required, a softenersuch as glycerol or sorbitol. The dry capsules may contain the activeingredient in the form of granules, for example with fillers, such aslactose, binders, such as starches, and/or lubricants, such as talc ormagnesium stearate, and where appropriate stabilizers. In soft capsules,the active ingredient may be present in dissolved or preferablysuspended form, whereby oily adjuvants such as fat oils, paraffin oil orliquid propylene glycols are added; stabilizers and/or antibacterialadditives may also be added. Dyes or pigments can be added to thetablets or dragee coatings, for example to improve identification or todistinguish different dosages of active ingredient.

[0113] The usage in the treatment of a proliferative disease with one ofthe crystal forms B and in particular A preferably takes place wherebythe crystal form (preferably as for the usage in the preparation of aninfusion solution, as described above) is administered to a warm-bloodedanimal, especially a human, in a dose which can be determined at between20 and 133%, preferably between 25 and 100%, of the Maximum ToleratedDose (MTD) by standard methods, for example using a modified Fibronacciseries, in which the increases in dosages for successive amounts are100%, 67%, 50% and 40% followed by 33% for all subsequent doses; and, ifnecessary, one or more further doses administered in the dosage rangegiven above for the first dose, each dose after a period of time whichallows sufficient recovery of the individual being treated after thepreceding administration, in particular one week or more after the firstadministration, preferably 2 to 10 weeks, especially 3 to 6 weeks aftereach preceding administration. In general, this treatment scheme, inwhich a high dosage is administered once, twice or several times withsufficiently long intervals between the individual administrations forrecovery to take place, is preferred over a more frequent treatment withlower doses, since hospitalisation is less frequent and for a shorterperiod and an improved anti-tumour effect can be expected. The dosage ofepothilone B for humans is preferably between 0.1 and 50 mg/m²,preferably between 0.2 and 10 mg/m².

[0114] The following Examples serve to illustrate the invention withoutlimiting its scope.

[0115] Caution: When handling epothilones, appropriate protectivemeasures must be taken, where necessary, in view of their high toxicity.

[0116] The 750 and 5000 litre fermenters used in the following arerefined steel fermenters from the company Alpha AG, Nidau, Switzerland.

EXAMPLE 1 Preparation of the Strain BCE33/10 and the Strain BCE63/114 byMeans of Mutation and Selection

[0117] The strain employed is the mutant BCE33/10 (deposited at theGerman Collection of Micro-organisms and Cell Cultures under number DSM11999 on Feb. 9, 1998) or the mutant BCE63/114 (deposited at the GermanCollection of Microorganisms and Cell Cultures under number DSM 12539 onNov. 27, 1998), which is derived from the strain Sorangium cellulosumSoce90 by mutation and selection as described below. In liquid media,the mutant BCE33/10, as well as BCE63/114, forms bacilli typical ofSorangia, with rounded ends and a length of 3-6 μm, as well as a widthof ca. 1 μm. Sorangium cellulosum Soce90 was obtained from the GermanCollection of Microorganisms under number DSM 6773.

[0118] Preparation of the mutant BCE33/10 comprises three mutation stepswith UV light and selections of individual colonies. The procedure indetail is carried out in accordance with the following operating steps

[0119] Cultivation from the ampoule: The cells of the DSM6773 ampouleare transferred to 10 ml of G52 medium in a 50 ml Erlenmeyer flask andincubated for 6 days in an agitator at 30° C. and at 180 rpm. 5 ml ofthis culture are transferred to 50 ml of G52 medium (in a 200 mlErlenmeyer flask) and incubated at 180 rpm for 3 days in an agitator at30° C.

[0120] First UV mutation step and selection: Portions of 0.1 ml of theabove culture are plated out onto several Petri dishes containing agarmedium S42. The plates are then each exposed to UV light (maximumradiation range of 250-300 nm) for 90 or 120 seconds at 500 μwatt percm². The plates are then incubated for 7-9 days at 30° C., untilindividual colonies of 1-2 mm are obtained. The cells of 100-150colonies are then each plated out from an individual colony by means ofa plastic loop in sectors onto Petri dishes containing S42 agar (4sectors per plate) and incubated for 7 days at 30° C. The cells thathave grown on an area of ca. 1 cm² agar surface are transferred by aplastic loop to 10 ml of G52 medium in a 50 ml Erlenmeyer flask andincubated for 7 days at 180 rpm in an agitator at 30° C. 5 ml of thisculture are transferred to 50 ml of G52 medium (in a 200 ml Erlenmeyerflask) and incubated at 180 rpm for 3 days in an agitator at 30° C. 10ml of this culture are transferred to 50 ml of 23B3 medium and incubatedfor 7 days at 180 rpm in an agitator at 30° C.

[0121] To determine the amounts of epothilone A and epothilone B formedin this culture, the following procedure is followed. The 50 ml culturesolution is filtered through a nylon sieve (150 μm pore size), and thepolystyrene resin Amberlite XAD16 retained on the sieve is rinsed with alittle water and subsequently added together with the filter to a 50 mlcentrifuge tube (Falcon Labware, Becton Dickinson AG Immengasse 7, 4056Basle). 10 ml of isopropanol (>99%) are added to the tube with thefilter. Afterwards, the well-sealed tube is shaken for 1 hour at 180 rpmin order dissolve the epothilone A and B, which is bonded to the resin,in the isopropanol. Subsequently, 1.5 ml of the liquid is centrifuged,and ca. 0.8 ml of the supernatant is added using a pipette to a HPLCtube. The HPLC analysis of these samples is effected as described belowunder HPLC analysis in the product analysis section. The HPLC analysisdetermines which culture contains the highest content of epothilone B.From the above-described sector plate of the corresponding colony (theplates have been stored at 4° C. in the meantime), cells from ca. 1 cm²of agar area are transferred by a plastic loop to 10 ml of G52 medium ina 50 ml Erlenmeyer flask and are incubated for 7 days at 180 rpm in anagitator at 30° C. 5 ml of this culture are transferred to 50 ml of G52medium (in a 200 ml Erlenmeyer flask) and incubated at 180 rpm for 3days in an agitator at 30° C.

[0122] Second and third UV mutation step and selection: The procedure isexactly the same as described above for the first UV mutation step,whereby the selected culture of the best colony from the first UVmutation is used for the second mutagenesis. For the third mutagenesis,the culture of the best colony from the second mutagenesis is usedaccordingly. The best colony after this third cycle of UV mutationsteps, followed by selection of the resulting strains for improvedepothilone B production, corresponds to mutant BCE33/10.

[0123] The strain BCE 63/114 is obtained from another (fourth) mutationstep from the strain BCE33/10, which is carried out in exactly the sameway as the above-mentioned mutation steps.

[0124] Preservation of the Strain

[0125] 10 ml of a 3 day old culture in G52 medium (50 ml medium in a 200ml Erlenmeyer flask, 30° C. and 180 rpm) are transferred to 50 ml of23B3 medium (in a 200 ml Erlenmeyer flask) and incubated for 3 days at180 rpm in an agitator at 30° C. 1 ml portions of this culture areremoved in a form which is as homogeneous as possible (prior to eachremoval the culture is shaken by hand in the Erlenmeyer flask) togetherwith the polystyrene resin Amberlite XAD16 (polystyrene adsorptionresin, Rohm & Haas, Frankfurt, Germany), then filled into 1.8 ml Nunccryotubes (A/S Nunc, DK 4000 Roslide, Denmark) and stored either at −70°C. or in liquid nitrogen.

[0126] Cultivation of the strains from these ampoules is effected byheating them in the air to room temperature, and subsequentlytransferring the entire content of the cryotube to 10 ml G52 medium inan 50 ml Erlenmeyer flask and incubating for 5-7 days at 180 rpm in anagitator at 30° C.

[0127] Media

[0128] G52 Medium: yeast extract, low in salt (Springer, Maison Alfort,France) 2 g/l MgSO₄ (7 H₂O) 1 g/l CaCl₂ (2 H₂O) 1 g/l soya meal defatted(Mucedola S.r.I., Settimo Milan, Italy) 2 g/l potato starch Noredux(Blattmann, Wädenswil, Switzerland) 8 g/l glucose anhydrous 2 g/lFe-EDTA 8 g/l (Product No. 03625, Fluka Chemie AG, CH) 1 ml/l pH 7.4,corrected with KOH Sterilisation: 20 mins. 120° C.

[0129] S42 Agar-Medium: as described S. Jaoua et al. Plasmid 28,157-165(1992) 23B3 Medium: glucose 2 g/l potato starch Noredux (Blattmann,Wädenswil, Switzerland) 20 g/l soya meal defatted (Mucedola S.r.I.,Settimo Milan, Italy) 16 g/l Fe-EDTA (Product No. 03625, Fluka, Buchs,Switzerland) 0.008 g/l HEPES Fluka, Buchs, Switzerland 5 g/l polystyreneresin XAD16 (Rohm and Haas) 2% v/v H₂O deionised correction of pH to 7.8with NaOH sterilisation for 20 mins. at 120° C.

[0130] (HEPES=4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid)

EXAMPLE 2 Cultivation in Order to Produce the Epothilones

[0131] Strain: Sorangium cellulosum Soce-90 BCE 33/10

Example 1

[0132] Preservation of the strain: In liquid N₂, as in Example 1. Media:Precultures and intermediate cultures: G52 Main culture: 1B12

[0133] G52 Medium: yeast extract, low in salt (BioSpringer, MaisonAlfort, France) 2 g/l MgSO₄ (7 H₂O) 1 g/l CaCl₂ (2 H₂O) 1 g/l soya mealdefatted Soyamine 50T (Lucas Meyer, Hamburg, 2 g/l Germany) potatostarch Noredux A-150 (Blattmann, Waedenswil, 8 g/l Switzerland) glucoseanhydrous 2 g/l EDTA-Fe(III)-Na salt (8 g/l) 1 ml/1 pH 7.4, correctedwith KOH Sterilisation: 20 mins. 120° C.

[0134] 1B12 Medium: 1B12 Medium: potato starch Noredux A-150 (Blattmann,Waedenswil, 20 g/l Switzerland) soya meal defatted Soyamine 50T (LucasMeyer, Hamburg, 11 g/l Germany) EDTA-Fe(III)-Na salt 8 mg/l pH 7.8,corrected with KOH Sterilisation: 20 mins. 120° C.

[0135] Addition of cyclodextrins and cyclodextrin derivatives:

[0136]  Cyclodextrins (Fluka, Buchs, Switzerland, or Wacker Chemie,Munich, Germany) in different concentrations are sterilised separatelyand added to the 1 B12 medium prior to seeding.

[0137] Cultivation: 1 ml of the suspension of Sorangium cellulosumSoce-90 BCE 33/10 from a liquid N₂ ampoule is transferred to 10 ml ofG52 medium (in a 50 ml Erlenmeyer flask) and incubated for 3 days at 180rpm in an agitator at 30° C., 25 mm displacement. 5 ml of this cultureis added to 45 ml of G52 medium (in a 200 ml Erlenmeyer flask) andincubated for 3 days at 180 rpm in an agitator at 30° C., 25 mmdisplacement. 50 ml of this culture is then added to 450 ml of G52medium (in a 2 litre Erlenmeyer flask) and incubated for 3 days at 180rpm in an agitator at 30° C., 50 mm displacement.

[0138] Maintenance culture: The culture is overseeded every 3-4 days, byadding 50 ml of culture to 450 ml of G52 medium (in a 2 litre Erlenmeyerflask). All experiments and fermentations are carried out by startingwith this maintenance culture.

[0139] Tests in a Flask:

[0140] (I) Preculture in an Agitating Flask:

[0141] Starting with the 500 ml of maintenance culture, 1×450 ml of G52medium are seeded with 50 ml of the maintenance culture and incubatedfor 4 days at 180 rpm in an agitator at 30° C., 50 mm displacement.

[0142] (ii) Main Culture in the Agitating Flask:

[0143] 40 ml of 1B12 medium plus 5 g/l 4-morpholine-propane-sulfonicacid (=MOPS) powder (in a 200 ml Erlenmeyer flask) are mixed with 5 mlof a 1 Ox concentrated cyclodextrin solution, seeded with 10 ml ofpreculture and incubated for 5 days at 180 rpm in an agitator at 30° C.,50 mm displacement.

[0144] Fermentation: Fermentations are carried out on a scale of 10litres, 100 litres and 500 litres. 20 litre and 100 litre fermentationsserve as an intermediate culture step. Whereas the precultures andintermediate cultures are seeded as the maintenance culture 10% (v/v),the main cultures are seeded with 20% (v/v) of the intermediate culture.Important: In contrast to the agitating cultures, the ingredients of themedia for the fermentation are calculated on the final culture volumeincluding the inoculum. If, for example, 18 litres of medium +2 litresof inoculum are combined, then substances for 20 litres are weighed in,but are only mixed with 18 litres!

[0145] Preculture in an Agitating Flask:

[0146] Starting with the 500 ml maintenance culture, 4×450 ml of G52medium (in a 2 litre Erlenmeyer flask) are each seeded with 50 mlthereof, and incubated for 4 days at 180 rpm in an agitator at 30° C.,50 mm displacement.

[0147] Intermediate Culture, 20 Litres or 100 Litres:

[0148] 20 litres: 18 litres of G52 medium in a fermenter having a totalvolume of 30 litres are seeded with 2 litres of the preculture.Cultivation lasts for 3-4 days, and the conditions are: 30° C., 250 rpm,0.5 litres air per litre liquid per min, 0.5 bars excess pressure, no pHcontrol.

[0149] 100 litres: 90 litres of G52 medium in a fermenter having a totalvolume of 150 litres are seeded with 10 litres of the 20 litreintermediate culture. Cultivation lasts for 3-4 days, and the conditionsare: 30° C., 150 rpm, 0.5 litres of air per litre liquid per min, 0.5bars excess pressure, no pH control.

[0150] Main Culture, 10 Litres, 100 Litres or 500 Litres:

[0151] 10 litres: The media substances for 10 litres of 1 B12 medium aresterilised in 7 litres of water, then 1 litre of a sterile 10%2-(hydroxypropyl)-β-cyclodextrin solution are added, and seeded with 2litres of a 20 litre intermediate culture. The duration of the mainculture is 6-7 days, and the conditions are: 30° C., 250 rpm, 0.5 litresof air per litre of liquid per min, 0.5 bars excess pressure, pH controlwith H₂SO₄/KOH to pH 7.6+/−0.5 (i.e. no control between pH 7.1 and 8.1).

[0152] 100 litres: The media substances for 100 litres of 1 B12 mediumare sterilised in 70 litres of water, then 10 litres of a sterile 10%2-(hydroxypropyl)-α-cyclodextrin solution are added, and seeded with 20litres of a 20 litre intermediate culture. The duration of the mainculture is 6-7 days, and the conditions are: 30° C., 200 rpm, 0.5 litresair per litre liquid per min., 0.5 bars excess pressure, pH control withH₂SO₄/KOH to pH 7.6+/−0.5. The chain of seeding for a 100 litrefermentation is shown schematically as follows:

[0153] 500 litres: The media substances for 500 litres of 1B12 mediumare sterilised in 350 litres of water, then 50 litres of a sterile 10%2-(hydroxypropyl)-β-cyclodextrin solution are added, and seeded with 100litres of a 100 litre intermediate culture. The duration of the mainculture is 6-7 days, and the conditions are: 30° C., 120 rpm, 0.5 litresair per litre liquid per min., 0.5 bars excess pressure, pH control withH₂SO₄/KOH to pH 7.6+/−0.5.

[0154] Product Analysis:

[0155] Preparation of the Sample:

[0156] 50 ml samples are mixed with 2 ml of polystyrene resin AmberliteXAD16 (Rohm+Haas, Frankfurt, Germany) and shaken at 180 rpm for one hourat 30° C. The resin is subsequently filtered using a 150 μm nylon sieve,washed with a little water and then added together with the filter to a15 ml Nunc tube.

[0157] Elution of the Product from the Resin:

[0158] 10 ml of isopropanol (>99%) are added to the tube with the filterand the resin. Afterwards, the sealed tube is shaken for 30 minutes atroom temperature on a Rota-Mixer (Labinco BV, Netherlands). Then, 2 mlof the liquid are centrifuged off and the supernatant is added using apipette to HPLC tubes.

[0159] HPLC Analysis: Column: Waters-Symetry C18, 100 × 4 mm, 3.5 μmWAT066220 + preliminary column 3.9 × 20 mm WAT054225 Solvents: A: 0.02%phosphoric acid B: Acetonitrile (HPLC-Quality) Gradient: 41% B from 0 to7 min. 100% B from 7.2 to 7.8 min. 41% B from 8 to 12 min. Oven temp.:30° C. Detection: 250 nm, UV-DAD detection Injection vol.: 10 μlRetention time: Epo A: 4.30 min Epo B: 5.38 min

EXAMPLE 2A Effect of the Addition of Cyclodextrin and CyclodextrinDerivatives to the Epothilone Concentrations Attained

[0160] All the cyclodextrin derivatives tested here come from thecompany Fluka, Buchs, CH. The tests are carried out in 200 ml agitatingflasks with 50 ml culture volume. As controls, flasks with adsorberresin Amberlite XAD-16 (Rohm & Haas, Frankfurt, Germany) and without anyadsorber addition are used. After incubation for 5 days, the followingepothilone titres can be determined by HPLC: TABLE 1 order Conc Epo AEpo B Addition No. [% w/v]¹ [mg/l] [mg/l] Amberlite XAD-16 (v/v) 2.0 9.23.8 (% v/v) 2-hydroxypropyl-β-cyclodextrin 56332 0.1 2.7 1.72-hydroxypropyl-β-cyclodextrin ″ 0.5 4.7 3.32-hydroxypropyl-β-cyclodextrin ″ 1.0 4.7 3.42-hydroxypropyl-β-cyclodextrin ″ 2.0 4.7 4.12-hydroxypropyl-β-cyclodextrin ″ 5.0 1.7 0.52-hydroxypropyl-α-cyclodextrin 56330 0.5 1.2 1.22-hydroxypropyl-α-cyclodextrin ″ 1.0 1.2 1.22-hydroxypropyl-α-cyclodextrin ″ 5.0 2.5 2.3 β-cyclodextrin 28707 0.11.6 1.3 β-cyclodextrin ″ 0.5 3.6 2.5 β-cyclodextrin ″ 1.0 4.8 3.7β-cyclodextrin ″ 2.0 4.8 2.9 β-cyclodextrin ″ 5.0 1.1 0.4methyl-β-cyclodextrin 66292 0.5 0.8 <0.3 methyl-β-cyclodextrin ″ 1.0<0.3 <0.3 methyl-β-cyclodextrin ″ 2.0 <0.3 <0.3 2,6di-o-methyl-β-cyclodextrin 39915 1.0 <0.3 <0.32-hydroxypropyl-γ-cyclodextrin 56334 0.1 0.3 <0.32-hydroxypropyl-γ-cyclodextrin ″ 0.5 0.9 0.82-hydroxypropyl-γ-cyclodextrin ″ 1.0 1.1 0.72-hydroxypropyl-γ-cyclodextrin ″ 2.0 2.6 0.72-hydroxypropyl-γ-cyclodextrin ″ 5.0 5.0 1.1 no addition 0.5 0.5

EXAMPLE 2B 10 Litre Fermentation with 1%2-(hydroxypropyl)-β-cyclodextrin)

[0161] Fermentation is carried out in a 15 litre glass fermenter. Themedium contains 10 g/l of 2-(hydroxypropyl)-α-cyclodextrin from WackerChemie, Munich, Germany. The progress of fermentation is illustrated inTable 2. Fermentation is ended after 6 days and working up takes place.TABLE 2 Progress of a 10 litre fermentation duration of culture [d] EpoA [mg/l] Epo B [mg/l] 0 0 0 1 0 0 2 0.5 0.3 3 1.8 2.5 4 3.0 5.1 5 3.75.9 6 3.6 5.7

EXAMPLE 2C 100 Litre Fermentation with 1%2-(hydroxypropyl)-β-cyclodextrin):

[0162] Fermentation is carried out in a 150 litre fermenter. The mediumcontains 10 g/l of 2-hydroxypropyl)-β-cyclodextrin. The progress offermentation is illustrated in Table 3. The fermentation is harvestedafter 7 days and worked up. TABLE 3 Progress of a 100 litre fermentationduration of culture [d] Epo A [mg/l] Epo B [mg/l] 0 0 0 1 0 0 2 0.3 0 30.9 1.1 4 1.5 2.3 5 1.6 3.3 6 1.8 3.7 7 1.8 3.5

EXAMPLE 2D 500 Litre Fermentation with 1%2-(hydroxypropyl)-β-cyclodextrin):

[0163] Fermentation is carried out in a 750 litre fermenter. The mediumcontains 10 g/l of 2-(hydroxypropyl)-β-cyclodextrin. The progress offermentation is illustrated in Table 4. The fermentation is harvestedafter 7 days and worked up. TABLE 4 Progress of a 500 litre fermentationduration of culture [d] Epo A [mg/l] Epo B [mg/l] 0 0 0 1 0 0 2 0 0 30.6 0.6 4 1.7 2.2 5 3.1 4.5 6 3.1 5.1

EXAMPLE 2E Comparison Example 10 litre fermentation without adding anadsorber

[0164] Fermentation is carried out in a 15 litre glass fermenter. Themedium does not contain any cyclodextrin or other adsorber. The progressof fermentation is illustrated in Table 5. The fermentation is notharvested and worked up. TABLE 5 Progress of a 10 litre fermentationwithout adsorber. Epothilone A Epothilone B duration of culture [d][mg/l] [mg/l] 0 0 0 1 0 0 2 0 0 3 0 0 4 0.7 0.7 5 0.7 1.0 6 0.8 1.3

EXAMPLE 3 Working up of the epothilones: Isolation from a 500 Litre MainCulture:

[0165] The volume of harvest from the 500 litre main culture of example2D is 450 litres and is separated using a Westfalia clarifying separatorType SA-20-06 (rpm=6500) into the liquid phase (centrofugate+rinisingwater=650 litres) and solid phase (cells=ca.15 kg). The main part of theepothilones are found in the centrifugate. The centrifuged cell pulpcontains <15% of the determined epothilone portion and is not furtherprocessed. The 650 litre centrifugate is then placed in a 4000 litrestirring vessel, mixed with 10 litres of Amberlite XAD-16(centrifugate:resin volume=65:1) and stirred. After a period of contactof ca. 2 hours, the resin is centrifuged away on a Heine overflowcentrifuge (basket content 40 litres; rpm=2800). The resin is dischargedfrom the centrifuge and washed with 10-15 litres of deionsed water.Desorption is effected by stirring the resin twice, each time inportions with 30 litres of isopropanol in 30 litre glass stirringvessels for 30 minutes. Separation of the isopropanol phase from theresin takes place using a suction filter. The isopropanol is thenremoved from the combined isopropanol phases by adding 15-20 litres ofwater in a vacuum-operated circulating evaporator (Schmid-Verdampfer)and the resulting water phase of ca. 10 litres is extracted 3× each timewith 10 litres of ethyl acetate. Extraction is effected in 30 litreglass stirring vessels. The ethyl acetate extract is concentrated to 3-5litres in a vacuum-operated circulating evaporator (Schmid-Verdampfer)and afterwards concentrated to dryness in a rotary evaporator (Büchitype) under vacuum. The result is an ethyl acetate extract of 50.2 g.The ethyl acetate extract is dissolved in 500 ml of methanol, theinsoluble portions filtered off using a folded filter, and the solutionadded to a 10 kg Sephadex LH 20 column (Pharmacia, Uppsala, Sweden)(column diameter 20 cm, filling level ca. 1.2 m). Elution is effectedwith methanol as eluant. Epothilone A and B is present predominantly infractions 21-23 (at a fraction size of 1 litre). These fractions areconcentrated to dryness in a vacuum on a rotary evaporator (total weight9.0 g). These Sephadex peak fractions (9.0 g) are thereafter dissolvedin 92 ml of acetonitrile:water:methylene chloride=50:40:2, the solutionfiltered through a folded filter and added to a RP column (equipmentPrepbar 200, Merck; 2.0 kg LiChrospher RP-18 Merck, grain size 12 μm,column diameter 10 cm, filling level 42 cm; Merck, Darmstadt, Germany).Elution is effected with acetonitrile:water=3:7 (flow rate=500 ml/min.;retention time of epothilone A=ca. 51-59 mins.; retention time ofepothilone B=ca. 60-69 mins.). Fractionation is monitored with a UVdetector at 250 nm. The fractions are concentrated to dryness undervacuum on a Büchi-Rotavapor rotary evaporator. The weight of theepothilone A peak fraction is 700 mg, and according to HPLC (externalstandard) it has a content of 75.1%. That of the epothilone B peakfraction is 1980 mg, and the content according to HPLC (externalstandard) is 86.6%. Finally, the epothilone A fraction (700 mg) iscrystallised from 5 ml of ethyl acetate:toluene=2:3, and yields 170 mgof epothilone A pure crystallisate [content according to HLPC (% ofarea)=94.3%]. Crystallisation of the epothilone B fraction (1980 mg) iseffected from 18 ml of methanol and yields 1440 mg of epothilone B purecrystallisate [content according to HPLC (%) of area=99.2%]. m.p.(Epothilone B): 124-125° C.; ¹H-NMR data for Epothilone B:

[0166] 500 MHz-NMR, solvent: DMSO-d6. Chemical displacement δ in ppmrelative to TMS. s=singlet; d=doublet; m=multiplet δ (Multiplicity)Integral (number of H) 7.34(s) 1 6.50(s) 1 5.28(d) 1 5.08(d) 1 4.46(d) 14.08(m) 1 3.47(m) 1 3.11(m) 1 2.83(dd) 1 2.64(s) 3 2.36(m) 2 2.09(s) 32.04(m) 1 1.83(m) 1 1.61(m) 1 1.47-1.24(m) 4 1.18(s) 6 1.13(m) 2 1.06(d)3 0.89(d + s, overlapping) 6 Σ = 41

[0167] In this example (Example 3), epothilone B is obtained in thecrystal modification A, which is characterised by the X-ray diffractiondiagram of modification A (see general part of the present disclosure).

EXAMPLE 4 Crystal Modification B of Epothilone B

[0168] 50 mg of epothilone B (obtained for example as above) aresuspended in 1 ml of isopropanol and shaken for 24 hours at 25° C. Theproduct is filtered and dried. After drying under a high vacuum,epothilones B are obtained in the form of white crystals. The crystalmodification of the product is characterised by the X-ray diffractiondiagram of modification B (see general part of the present disclosure).

EXAMPLE 5 3000 Litre Fermentation with 2-(hydroxypropyl)-β-cyclodextrin)

[0169] Fermentation is carried out with the strain BCE 63/114 in a 5000litre fermenter in 1B12 medium (filled volume 3000 litres).

[0170] Maintenance culture: Preparation is effected as described inExample 1 (strain preservation) and 2 for the strain BCE33/10, but usinginstead the strain BCE63/114.

[0171] Precultures:

[0172] Preparation of the precultures is effected analogously to Example2 (ii), but with the following precultures and with strain BCE 63/114:

[0173] From a 500 ml maintenance culture [as described in example 2(ii)], 50 ml portions are placed in 4 Erlenmeyer flasks, thus producingfour 500 ml precultures in G52 medium for 3 days at 30° C. and at 180rpm. These 4 precultures (2 litres) are then used for three intermediatecultures each of 20 litres (G52 medium, 4 days, 30° C., 250 rpm). 5litre portions of these intermediate cultures are used to produce three50 litre intermediate cultures (G52 medium, 3 days, 30° C., 200 rpm). 50litres of these 50 litre intermediate cultures are used twice to growtwo 600 litre intermediate cultures (G52 medium, 4 days, 30° C., 120rpm).

[0174] Main culture: The media substances for 3000 litres are sterilisedin 2100 litres of water, then 300 litres of a sterile 10%2-(hydroxypropyl)-β-cyclodextrin solution are added, and seeded with 600litres of an intermediate culture. The duration of the main culture is6-7 days, and the conditions are: 30° C., 100 rpm, 0.5 litres of air perlitre liquid per minute, 0.5 bars excess pressure, pH control withH₂SO₄/KOH to pH 7.6+/−0.5 (i.e. no control between pH 7.1 and 8.1). Theprogress of fermentation is illustrated in Table 6. TABLE 6 Progress ofa 3000 litre fermentation duration of culture [d] Epothilone A [mg/l]Epothilone B [mg/l] 0 0 0 1 0 0 2 0 0 3 2.1 1.6 4 4.1 2.9 5 5.2 3.8 65.5 4.3

[0175] Working Up and Isolation of the Epothilones from a 3000 LitreMain Culture: (I) Resin Binding, Desorption and Extraction of theEpothilones (Ethyl Acetate Extract)

[0176] The volume of harvest from the 3000 litre main culture is 2900litres and is separated using a Westfalia clarifying separator TypeSA-20-06 (rpm=6500, flow rate 1400 litres/hour) into the liquid phase(centrfugate+rinsing water=2750 litres) and solid phase (cells=ca. 260kg). The main part of the epothilones are found in the centrifugate, Thecentrifuged cell pulp contains <15% of the determined epothilone portionand is not further processed. The 2750 litre centrifugate is then placedin a 4000 litre steel stirring vessel, mixed with 60 litres of AmberliteXAD-16 (centrifugate:resin volume=46:1) and stirred. After a period ofcontact of 16-20 hours, the resin is centrifuged away in a Heineoverflow centrifuge (basket content 40 litres; rpm=2800). The centrifugeis emptied by rinsing the basket content with deionised water when thecentrifuge is stationary. The XAD-16/deionised water slurry isthereafter freed from water on a suction filter (Ø 50 cm) and the resinwashed with 30 litres of deionised water. Desorption of the resin iseffected by stirring it in a 1600 litre stirring vessel twice, each timewith 220 litres of isopropanol for 30 minutes. Separation of theisopropanol phase from the resin takes place using a suction filter (Ø50 cm). The isopropanol is then removed from the isopropanol phase byadding 240-260 litres of water in a vacuum-operated circulatingevaporator (Schmid-Verdampfer) and the resulting water phase of ca. 125litres is extracted 3× each time with 100-125 litres of ethyl acetate.Extraction is effected in 1600 litre steel stirring vessels. The ethylacetate extracts are combined, concentrated to 3-5 litres invacuum-operated circulating evaporators (BüchiVerdampfer/Schmid-Verdampfer) and afterwards concentrated to dryness ina rotary evaporator (Büchi type) under vacuum. An ethyl acetate extractof 590 g is obtained.

[0177] HPLC-Purification of the Ethyl Acetate Extract (Separation ofEpothilones A and B)

[0178] Ca. 300 g of the above-mentioned ethyl acetate extract (with acontent of ca. 1-1.5% epothilone B) are suspended in 1.5 litres ofacetonitrile/water=3/1 (v/v), the solution is filtered through a foldedfilter and the filtrate added to a C-18 RP column [Prochrom apparatuswith 30 cm internal column diameter (Prochrom, Champigneulles, France)25 kg YMC gel, ODS-A, 120 Angstroem pore diameter, 5-15 μm grain size,spherical]. Elution is effected with acetonitrile/water=4/6 (v/v) at aflow rate of 2300 ml/min. Fractionation is monitored by means of on-lineHPLC [rapid HPLC at high temperatures (ca. 80° C.), on a shortseparating column (4.6 mm internal diameter x 75 mm length) and verysmall RP-18 particles (3.5 μm spherical), typical analysis times are <1minute, detection at 250 nm]. The valuable fractions (those with onlyepothilone B) are combined, the acetonitrile removed by distillation andthe aqueous phase extracted twice with isopropyl acetate. The organicphases are concentrated by distillation and the isopropyl acetateextract is obtained as the residue of evaporation.

[0179] (iii) Silica Gel Filtration of the Isopropyl Acetate Extract

[0180] Ca. 10 g of the combined residues of evaporation thus obtained(with a content of ca. 23% of epothilone B) are dissolved at roomtemperature in 360 ml of ethyl acetate, the solution is filtered througha folded filter and added to a column of silica gel (Prochrom apparatuswith 10 cm internal column diameter, 1.5 kg ICN 18-32 μm). Elution iseffected with ethyl acetate/n-hexane=4/1 (v/v) at a flow rate of 250ml/min at room temperature. Fractionation is monitored with a UVdetector at 250 nm. The valuable fractions are combined, the solventremoved by distillation and concentrated to dryness.

[0181] (iv) Extraction of the Pure Epothilones

[0182] Ca. 48 g of the combined residues obtained under (iii) (with acontent of ca. 90% of epothilone B) are dissolved in 1150 ml ofmethanol, mixed with 14.5 g of activated carbon and subsequentlyfiltered through a folded filter. The clear filtrate is subsequentlyconcentrated to dryness and the residue is recrystallised from 317 ml ofmethanol. 29.5 g of epothilone B are obtained in a purity of 99.7%, andwith a melting point of 124° C.

EXAMPLE 6 Infusion Concentrate

[0183] By dissolving in polyethylene glycol PEG 300, crystalmodification A of epothilone B, or crystal modification B of epothiloneB, is produced in a preconcentrate to produce infusion solutions, andstored in vials.

1. A process for concentrating epothilones in a culture medium for thebiotechnological preparation of these compounds, the process comprisingmicroorganisms which produce these compounds, whereby a complex-formingcomponent which is soluble in the culture medium is added to the medium.2. A process according to claim 1 for concentrating epothilones in aculture medium for the biotechnological preparation of these compounds,the process comprising myxobacteria as producers of natural substances,whereby a complex-forming component which is soluble in the culturemedium is added to the medium.
 3. A process according to claim 1 forconcentrating epothilones, in which a culture medium is used for thebiotechnological preparation of epothilones, the medium containing aSorangium strain suitable for the preparation thereof, water and othersuitable customary components of culture media, wherein one or morecyclodextrins are added to the medium, the cyclodextrins being selectedfrom α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, δ-cyclodextrin,ε-cyclodextrin, ζ-cyclodextrin, η-cyclodextrin and θ-cyclodextrin; or acyclodextrin derivative or a mixture of cyclodextrin derivativesselected from derivatives of a cyclodextrin in which one or more up toall of the hydroxy groups are etherified to an alkyl ether, anaryl-hydroxyalkyl ether; a hydroxyalkyl ether; a carboxyalkyl ether; aderivatised carboxy lower alkyl ether in which the derivatised carboxyis aminocarbonyl, mono- or di-lower-alkylaminocarbonyl, morpholino-,piperidino-, pyrrolidino- or piperazino-carbonyl, or alkyloxycarbonyl; asulfoalkyl ether; a cyclodextrin in which one or more OH groups areetherified with a radical of formula —O-[alk-O—]_(n)—H  wherein alk isalkyl and n is a whole number from and including 2 up to and including12; a cyclodextrin in which one or more OH groups are etherified with aradical of formula

 wherein R′ is hydrogen, hydroxy, —O-(alk-O)_(n)—H,—O-(alk(-R)—O—)_(p)—H or —O-(alk(-R)—O—)_(q)-alk-CO—Y; alk in all casesis alkyl; m, n, p, q and z are a whole number from 1 to 12; and Y is OR₁or NR₂R₃, wherein R₁, R₂ and R₃, independently of one another, arehydrogen or lower alkyl, or R₂ and R₃, combined with the bindingnitrogen signify morpholino, piperidino, pyrrolidino or piperazino; or abranched cyclodextrin in which etherifications or acetals exist withother sugar molecules, and which are selected from glucosyl-,diglucosyl-(G₂-β-cyclodextrin), maltosyl- and dimaltosyl-cyclodextrin,or N-acetylglucosaminyl-, glucosaminyl-, N-acetylgalactosaminyl- andgalactosaminyl-cyclodextrin; and a lower alkanoyl-, such as acetylesterof a cyclodextrin; or mixtures of two or more of the said cyclodextrinsand/or cyclodextrin derivatives.
 4. Process according to claim 3, inwhich the cyclodextrin and/or the cyclodextrin derivative is added tothe culture medium in a concentration of between 0.05 and 10 percent byweight (w/v).
 5. Process according to claim 4, in which the cyclodextrinand/or the cyclodextrin derivative is added in a concentration ofbetween 0.1 and 2 percent by weight.
 6. A process according to claim 3,in which the cyclodextrin derivative is selected from a cyclodextrin anda hydroxy lower alkyl cyclodextrin; or mixtures of one or more thereof.7. A process according to claim 1, in which the complex-formingcomponent is 2-hydroxy-propyl-β-cyclodextrin.
 8. A culture medium whichcomprises a complex-forming component and a microorganism suitable forthe production of epothilones.
 9. A culture medium according to claim 8,in which the complex-forming component used is a cyclodextrin, acyclodextrin derivative or a mixture of two or more complex-formingcomponents selected from cyclodextrins and cyclodextrin derivatives. 10.A culture medium according to claim 9, in which the microorganism is amyxobacterium.
 11. A process for the production of epothilones, in whichthe epothilones are obtained by working up a culture medium for thebiotechnological preparation of these compounds, the medium comprisingmyxobacteria as producers of natural substances, to which medium isadded a complex-forming component which is soluble in the culturemedium, and subsequently purifying and, if desired, separating theepothilones from one another.
 12. A process according to claim 11,wherein the epothilones are obtained by working up a culture medium forthe biotechnological preparation of these compounds, the culture mediumcontaining a myxobacterium of the genus Sorangium, and to which mediumis added a complex-forming component which is soluble in the culturemedium, the culture is separated into the solid and the liquid phase(centrifugate) by centrifugation; the centrifugate is mixed with a resinor is run over a column filled with such a resin; if necessary the resinis washed with water; the epothilone(s) is or are desorbed from theresin with a polar solvent; if necessary this is concentrated withprior, simultaneous or subsequent addition of water; an organic solventwhich is immiscible with water is added and the epothilone(s) is or aretransferred into the organic phase; the organic phase obtained isconcentrated if required; the epothilones from the organic solutionobtained are concentrated through a molecular sieve for compounds of lowmolecular weight; and subsequently the fractions containing theepothilones undergo separation on a reversed-phase column; wherebyepothilones A and B are obtained separately and, if desired, can befurther concentrated by recrystallisation.
 13. A process according toclaim 11 for the production of epothilone A and/or B, wherein theepothilones are obtained by working up a culture medium for thebiotechnological preparation of these compounds, to which medium isadded a complex-forming component which is soluble in the culturemedium, the culture is separated into the solid and the liquid phase(centrifugate) by centrifugation; the centrifugate is mixed with a resinor is run through a column filled with such a resin; if necessary theresin is washed with water; the epothilone(s) is or are desorbed fromthe resin with a polar solvent; if necessary the polar solvent isremoved with prior, simultaneous or subsequent addition of water; theresulting water phase is extracted with a solvent suitable for forming asecond phase; the organic phase obtained is concentrated if required,preferably to dryness; epothilone A and epothilone B are separated fromone another directly by reversed-phase chromatography whilst elutingwith an eluant containing a nitrile; subsequently concentrated; ifdesired, the residue is treated from an aqueous solution by extractingonce or several times with a solvent which is immiscible with water;dissolved in an appropriate solvent, filtered if necessary, added to asilica gel column and eluted with an appropriate solvent or solventmixture; and subsequently each fraction containing epothilone A or inparticular B is separately combined and concentrated by removing thesolvent; then the residue is dissolved in an appropriate alcohol, ifdesired, in order to obtain especially high purity, mixed with activatedcarbon and then filtered; and finally epothilone A or B is obtained byrecrystallisation.
 14. A process for the production of epothilones,which a) is a process for concentrating epothilones in a culture mediumfor the biotechnological preparation of these compounds, which containsa microorganism suitable for the preparation thereof, water and othersuitable customary constituents of culture media, whereby a cyclodextrinor a cyclodextrin derivative is added to the medium, or a mixture of twoor more of these compounds; and b) comprises a step for separatingepothilones from one another, which is characterised by chromatographyon a reversed-phase column with an eluant containing a loweralkyl-cyanide, whereby chromatography is carried out on column materialcharged with hydrocarbon chains, and an eluant containing a loweralkylnitrile is used; whereby if desired further working up steps andpurification steps are possible.
 15. A method of separating epothilonesfrom one another, which is characterised by chromatography on areversed-phase column with an eluant containing a lower alkyl-cyanide.16. A method according to claim 15, wherein a column material is usedwhich is charged with hydrocarbon chains containing 18 carbon atoms, andthe eluant used is a mixture of water and acetonitrile.
 17. A strain ofSorangium cellulosum, obtained by mutagenesis, which under otherwiseidentical conditions, produces more epothilones than Sorangiumcellulosum Soce90.
 18. A strain according to claim 17, selected from thestrains having the references BCE33/10 and BCE63/114.
 19. A crystal formof epothilone B having the reference modification A, which ischaracterised by the X-ray diffraction diagram reproduced in the form ofa table, obtained using a diffractometer with Cu-Kα₁ radiation. 2θIntensity 7.7 very strong 10.6 weak 13.6 average 14.4 average 15.5average 16.4 weak 16.8 weak 17.1 weak 17.3 weak 17.7 weak 18.5 weak 20.7strong 21.2 strong 21.9 weak 22.4 weak 23.3 strong 25.9 average 31.2weak 32.0 average


20. A crystal form of epothilone B having the reference modification B,which is characterised by the X-ray diffraction diagram reproduced inthe form of a table, obtained using a diffractometer with Cu-Kα₁radiation. 2θ Intensity 6.9 very strong 8.0 weak 8.3 average 10.8 strong11.5 average 12.4 weak 13.1 strong 15.5 weak 16.2 weak 16.7 average 18.1average 18.6 average 20.4 weak 20.9 strong 21.3 weak 21.5 very weak 22.5average 24.2 weak 25.1 average


21. A pharmaceutical composition which is suitable for administration toa warm-blooded animal for the treatment of a proliferative disease,which contains a quantity of an active ingredient according to one ofclaims 19 and 20, which is suitable for the treatment of said diseasetogether with a pharmaceutically acceptable carrier.
 22. Method oftreating a warm-blooded animal suffering from a proliferative disease,by administering a dosage of epothilone B which is effective fortreating said disease according to one of claims 19 and 20 to awarm-blooded animal requiring such treatment.
 23. Use of a new crystalform of epothilone B according to one of claims 19 and 20 in theproduction of pharmaceutical preparations.